Insulin regulation of lipoprotein lipase (LPL) in fat cells was studied in fully differentiated 3T3-L1 adipocytes. Insulin (less than 10 to the minus 7th power M) elicited a rapid increase (10x) in release of LPL from cells to medium, and a slower increase (5x) in total LPL in the cells. The insulin-induced increase in LPL activity in cells required glucose. Glucose added to cells incubated 20 hr with insulin in hexose-free medium elicited a full response to insulin in 2 hr. The latter was prevented by cycloheximide but not actinomycin D, whereas the response in cells incubated with glucose for 24 hr was blocked completely by either inhibitor. It is concluded insulin regulates LPL through three major actions: 1) It stimulates release of LPL from fat cells. 2) It stimulates gene expression and RNA synthesis independently of effects on hexose and amino acid transport. 3) It stimulates hexose transport which enhances expression of nuclear effects of insulin at the translational level.